1. Field of the Invention
The present invention relates to chemiluminescent systems, especially enhanced chemiluminescent systems for assay of peroxidase activity. For the purposes of this specification, xe2x80x9cenhanced chemiluminescencexe2x80x9d is defined by the fact that the total light output of the present invention and/or signal to noise ratio of the present invention is greater than the output given by a 2,3-dihydro-1,4-phthalazinedione/oxidizing agent/peroxidase solution in the absence of the luminescent enhancer. When a 2,3-dihydroxy-1,4-phthalazine dione is used in the xe2x80x9cenhanced chemiluminescencexe2x80x9d system, it may most accurately be compared to the same compound in the absence of a luminescent enhancer for comparison under this definition. More particularly, the present invention relates to such systems wherein increased light output is obtained, increased rates of attaining maximum light output may be obtained, and extended duration of light output can be achieved. In combination, these characteristics of the present invention achieve lower limits of detection and more useful assays. These improvements are due to the use of a single azine enhancer containing a water-soluble moiety. The invention also relates to the use of highly purified enhancers which significantly improve various properties associated with the chemiluminescence developed during the peroxidase catalyzed oxidation of a chemiluminescent compound such as luminol, isoluminol and their derivatives, such as chemiluminescent cyclic diacylhydrazides generally. The invention is particularly applicable to detection of proteins, nucleic acids and other analytes using all types of membrane-based assays such as dot blotting, western blotting, southern blotting, and northern blotting, etc. Furthermore, the invention is particularly applicable to the detection of proteins, nucleic acids, and other analytes using all types of solution-based, luminometric assays such as ELISAs (Enzyme Linked Immunoabsorbent Assays), coated tube assays, bead assays, etc.
2. Background of the Invention
Chemiluminescent detection of molecules of synthetic or natural origin such as proteins and nucleic acids (DNA and RNA), as well as other biologic molecules, is increasingly replacing radioactive detection as the method of choice where sensitivity is critical. In such assays, luminescence is customarily achieved by the oxidation of a luminol or isoluminol substrate in the presence of an oxidizing agent such as hydrogen peroxide or hydrogen peroxide source, such as perborate, and a peroxidase catalyst such as horseradish peroxidase.
To obtain useful levels of luminescence (e.g., detectable levels) by customary techniques, a luminescent enhancer is also present during oxidation. Among the enhancers which have been successfully used with peroxidases are aromatic amines (U.S. Pat. No. 4,729,950), phenols (U.S. Pat. No. 4,598,044), and azines (e.g., phenothiazines, phenoxazines) and phenolindophenols (U.S. Pat. No. 5,171,668).
Currently, in chemiluminescent assays for proteins, the assay system of choice is a horseradish peroxidase catalyst, a phenolic enhancer, and a hydrogen peroxide source. The peroxidase catalyst is generally coupled, directly or indirectly, to a ligand, (e.g., an antibody, having binding specificity for the protein of interest). In turn, the intensity of luminescent response from the oxidation of the substrate (e.g., the chemiluminescent cyclic diacylhydrazide, such as the luminol or isoluminol derivative) by the bound peroxidase catalyst in the presence of an enhancer is used as a measurement of the amount of protein.
Nucleic acids can be assayed in a similar manner to proteins. However, with nucleic acids the current chemiluminescent system of choice is an alkaline phosphatase catalyst and a phospho-substituted dioxetane substrate. In such systems, the light output is improved by the presence of certain flouorophore-containing micelles (Focus 12, Number 1, pp. 9-12).
While the above-described assays for proteins and nucleic acids are currently in use, their use is accompanied by certain drawbacks. For example, while chemiluminescent systems for assaying proteins using peroxidase can yield rapid luminescence, such luminescence is short lived and of modest intensity, and has significant limits in its ability to detect small amounts of the target analyte. Accordingly, a rapid measurement of luminescence intensity must be made and such a rapid measurement may not always be possible or desirable. Similarly, there are several limitations to the current chemiluminescent systems used for assaying nucleic acids. First, the alkaline phosphatase procedures are more expensive than the peroxidase catalyzed systems. In addition, the luminescence achieved is slow to develop, even though once developed, the luminescence continues for an extended period. Therefore, assays of long duration are required to achieve acceptable levels of sensitivity.
Now, however, in accordance with the present invention, there is provided an improved method for the chemiluminescent assay of peroxidase activity which is generally useful in connection with the detection of analytes of all types (e.g., biological macromolecules, organic molecules, natural or synthetic molecules, etc). The invention is particularly applicable to detection of proteins and nucleic acids using all types of membrane-based assays by techniques such as dot blotting, western, blotting, southern blotting, and northern blotting, colony filter hybridization, etc. Furthermore, the invention is particularly applicable to the detection of analytes using all types of solution-based, luminometric assays, such as ELISAs (Enzyme Linked Immunoabsorbent Assays), coated tube assays, bead assays, etc..
In the present specification, an unprecedented degree of luminescence is developed more rapidly than previously reported, and the intense luminescence persists for a period of time comparable to the dioxetanes (which is much longer than previously reported for peroxidase-based systems). Thus, by using the method of the present invention rapid development of a high intensity luminescence is achieved and said luminescence is of an extended duration. Thus, this method combines the advantageous features of both of the current chemiluminescent methodologies (those based on peroxidase or alkaline phosphatase catalysts). Moreover, by combining the features of high light output with extended duration, unprecedented levels of sensitivity are achieved in many assay systems.
In one of its aspects the present invention provides an improvement in the method for the chemiluminescent assay of peroxidase activity which involves oxidizing a substrate (e.g., 2,3-dihydro-1,4-phthalazinedione, chemiluminescent cyclic diacylhydrazide, luminol, isoluminol, or other derivatives) in a solution containing the substrate, the catalyst (e.g., peroxidase), an oxidizing agent (e.g., hydrogen peroxide or hydrogen peroxide source) and an enhancer (e.g., an azine such as a phenothiazine or phenoxazine). The improvement resides in the use, as the enhancer, of azines such as the phenothiazines or phenoxazines free of specific classes of compounds which adversely affect the performance of the entire system, and which have been found to be present in previous enhancer compositions. These compounds, which must be reduced in concentration from their levels in conventional enhancer preparations are azines in which the nitrogen atom of the azine ring has a hydrogen bonded directly thereto. It is also preferred to provide aqueous solutions of the substrate, oxidizing agent, and enhancer by using enhancers which contain a water-solubilizing substituent such as alkyl sulfonates salts, substituted ammonium salts, or phosphonium salts. A further limited aspect of the present invention resides in providing an aqueous solution useful for assaying peroxidase activity which consists essentially of a chemiluminescent cyclic diacylhydrazide, an oxidizing agent, and an enhancer containing a water solubilizing